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Retrovirology BioMed Central Open Access Research Human T-cell leukemia virus type post-transcriptional control protein p28 is required for viral infectivity and persistence in vivo Brenda Yamamoto1,2,3, Min Li1,2, Matthew Kesic1,2, Ihab Younis1,2, Michael D Lairmore1,2,3,4 and Patrick L Green*1,2,3,4 Address: 1Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210, USA, 2Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA, 3Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State, University, Columbus, OH 43210, USA and 4Comprehensive Cancer Center and Solove Research Institute, The Ohio State University, Columbus, OH 43210, USA Email: Brenda Yamamoto - yamamoto.26@osu.edu; Min Li - li.583@osu.edu; Matthew Kesic - kesic.1@osu.edu; Ihab Younis - younis@mail.med.upenn.edu; Michael D Lairmore - lairmore.1@osu.edu; Patrick L Green* - green.466@osu.edu * Corresponding author Published: 12 May 2008 Retrovirology 2008, 5:38 doi:10.1186/1742-4690-5-38 Received: April 2008 Accepted: 12 May 2008 This article is available from: http://www.retrovirology.com/content/5/1/38 © 2008 Yamamoto et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Abstract Background: Human T-cell leukemia virus (HTLV) type and type are related but distinct pathogenic complex retroviruses HTLV-1 is associated with adult T-cell leukemia and a variety of immune-mediated disorders including the chronic neurological disease termed HTLV-1-associated myelopathy/tropical spastic paraparesis In contrast, HTLV-2 displays distinct biological differences and is much less pathogenic, with only a few reported cases of leukemia and neurological disease associated with infection In addition to the structural and enzymatic proteins, HTLV encodes regulatory (Tax and Rex) and accessory proteins Tax and Rex positively regulate virus production and are critical for efficient viral replication and pathogenesis Using an over-expression system approach, we recently reported that the accessory gene product of the HTLV-1 and HTLV-2 open reading frame (ORF) II (p30 and p28, respectively) acts as a negative regulator of both Tax and Rex by binding to and retaining their mRNA in the nucleus, leading to reduced protein expression and virion production Further characterization revealed that p28 was distinct from p30 in that it was devoid of major transcriptional modulating activity, suggesting potentially divergent functions that may be responsible for the distinct pathobiologies of HTLV-1 and HTLV-2 Results: In this study, we investigated the functional significance of p28 in HTLV-2 infection, proliferation, and immortaliztion of primary T-cells in culture, and viral survival in an infectious rabbit animal model An HTLV-2 p28 knockout virus (HTLV-2Δp28) was generated and evaluated Infectivity and immortalization capacity of HTLV-2Δp28 in vitro was indistinguishable from wild type HTLV-2 In contrast, we showed that viral replication was severely attenuated in rabbits inoculated with HTLV-2Δp28 and the mutant virus failed to establish persistent infection Conclusion: We provide direct evidence that p28 is dispensable for viral replication and cellular immortalization of primary T-lymphocytes in cell culture However, our data indicate that p28 function is critical for viral survival in vivo Our results are consistent with the hypothesis that p28 repression of Tax and Rex-mediated viral gene expression may facilitate survival of these cells by down-modulating overall viral gene expression Page of 11 (page number not for citation purposes) Retrovirology 2008, 5:38 Background The human T-cell leukemia viruses (HTLV types 1–4) are classified as complex retroviruses and members of the genus Deltaretrovirus [1] HTLV-1 and HTLV-2 infections are the most prevalent worldwide, whereas infections with HTLV-3 and HTLV-4 were discovered only recently in a very limited number of individuals in Africa [2,3] Although people infected with HTLV have a persistent antiviral immune response, these patients fail to clear virally infected cells A small percentage of HTLV-1infected individuals develop adult T-cell leukemia (ATL), a CD4+ lymphocyte malignancy, and various lymphocyte-mediated inflammatory diseases such as HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) [4-7] However, only a few cases of atypical hairy cell leukemia or neurologic disease have been associated with HTLV-2 infection [8-12] HTLV-1 and HTLV-2 have the capacity to promote T-lymphocyte growth both in cell culture and in infected individuals; however, the mechanism by which the virus persists in the infected individual, ultimately resulting in the oncogenic transformation of T-lymphocytes, is not completely understood In addition to the gag, pol, and env genes that encode the structural and enzymatic proteins, HTLV encodes tax/rex and accessory genes from pX open reading frames (ORFs) located in the 3' region of the genome Tax increases the rate of transcription from the viral long terminal repeat (LTR) [13-15] and modulates the transcription or activity of numerous cellular genes involved in cell growth and differentiation, cell cycle control, and DNA repair [16-20] Compelling evidence indicates that the pleiotropic effects of Tax on cellular processes are required for the transforming or oncogenic capacity of HTLV [21-23] Rex acts posttranscriptionally by preferentially binding, stabilizing and selectively exporting the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm, thus controlling the expression of the structural and enzymatic proteins as well as virion production [24-26] Although both Tax and Rex are key positive regulators essential for efficient viral replication and, ultimately, cellular transformation, it has been hypothesized that the unregulated expression of these genes would result in the death of the infected cell in vivo via the induction of apoptosis and/or host immune response Growing evidence indicates that the HTLV-1 p30 and the HTLV-2 p28 accessory proteins encoded by pX ORF II regulate HTLV gene expression and therefore may contribute to the pathobiology of the virus The homology between p30 and p28 is limited with the N-terminal 49 amino acids of p28 sharing 77% identity with the C-terminal portion of p30 [27,28] Using over-expression studies, we and others reported that the nuclear/nucleolar-localizing p30 or p28 (p30/p28) specifically bind to and retain tax/ http://www.retrovirology.com/content/5/1/38 rex mRNA in the nucleus [29,30] Furthermore, inhibition of tax/rex mRNA export by p30/p28 appears to be co-transcriptional and requires an interaction between p30/p28 and Tax complexes on the viral promoter, which facilitates the co-migration of p30/p28 with RNA pol II until the protein encounters the newly synthesized downstream RNA binding sequence [31] In addition, Sinha-Datta et al demonstrated that p30 and Rex form a ribonucleoprotein ternary complex specifically on the tax/rex mRNA, which is consistent with its selective nuclear retention [32] Interestingly, p30 also has been shown to interact with transcriptional co-activators/acetyltransferases, p300/CBP and TIP60, displaying both positive and inhibitory transcriptional effects on viral and cellular promoters [33-37] Unlike p30, p28 does not display any significant transcriptional regulatory activity [29-31] suggesting the possibility of distinct or additional functions Together, these findings suggest that p30/p28 facilitates virus and/or infected cell survival by regulating viral gene expression Under standard cell culture conditions, p30 was dispensable for viral infection, replication and immortalization of T-lymphocytes in vitro [38] In vivo studies using a rabbit model of infection have revealed that p30 is important for the establishment of persistent infection [39,40] However, more recent identification of HTLV-1 Hbz, found on the opposite coding strand partially overlapping p30, makes precise interpretation of these studies difficult HTLV-2 containing a large deletion of the 3' proximal pX region maintained the capacity to efficiently replicate in and transform primary T-lymphocytes in culture, but was significantly attenuated in inoculated rabbits [41,42] However, the specific contribution of the HTLV-2 accessory gene products, particularly p28, to overall virus biology has not been determined In this study, we evaluated the functional role of p28 in the context of an HTLV-2 infectious molecular clone and determined its contribution to viral replication and viralinduced immortalization in cell culture as well as viral replication kinetics and persistence in inoculated rabbits Our findings indicate that the loss of p28 and thus its documented repressive post-transcriptional regulatory effect on Tax/Rex was not sufficient to disrupt the capacity of the virus to immortalize primary T-lymphocytes in culture However, in the in vivo rabbit infection model, a p28defective HTLV-2 had reduced replication and ability to establish persistent infection These results suggest that the posttranscriptional repression of retroviral gene expression by p28 down-modulates viral replication thereby directly affecting cell signaling and survival In addition, p28 may facilitate immune escape by HTLV infected cells by preventing their recognition by the host immune response Page of 11 (page number not for citation purposes) Retrovirology 2008, 5:38 http://www.retrovirology.com/content/5/1/38 Results Generation and characterization of the HTLV-2 p28 knockout mutant As a result of alternative splicing, HTLV-2 p28 has the potential to be expressed from two distinct singly-spliced mRNAs (Fig 1) Both mRNAs also have the potential to produce the amino terminal truncated p22/p20 Rex proteins [28,43] It is important to note that the p28 ORF has complete overlap with Tax exon and partial overlap with Rex exon (Fig 1) Using an over-expression system approach, previous studies revealed that p28 is at least in part functionally homologous to HTLV-1 p30 and has the capacity to specifically retain tax/rex mRNA in the nucleus, thus decreasing Tax and Rex protein and viral replication via a posttranscriptional mechanism [30] However, the specific role of p28 in the context of a proviral clone, and ultimately on virus biology, has not been investigated In order to determine the potential role of p28 in HTLV-2mediated cellular immortalization in cell culture and viral persistence in inoculated rabbits, a p28-deficient proviral clone (HTLV-2Δp28) was generated from the HTLV-2 molecular clone pH6neo To construct HTLV-2Δp28, a single nucleotide was altered by site directed mutagenesis, which introduced a stop codon at amino acid of the p28 ORF and had no affect on the overlapping Tax and Rex amino acid sequence We initially determined whether knocking out p28 altered Tax and/or Rex activities Cotransfection of wild-type HTLV-2 or HTLV-2Δp28, as a source of Tax, and the LTR-2-Luc reporter revealed that HTLV-2Δp28 had a consistently lower, but not signifi- 5’LTR 1Kb 2Kb gag 3Kb 4Kb 5Kb cantly different LTR-directed gene expression (Fig 2A) Moreover, cells transfected with HTLV-2Δp28 produced levels of p19 Gag in the culture supernatant similar to wild-type HTLV-2, indicating no significant repression of Rex function (Fig 2B) Based on the reported functional activity of over-expressed p28, we were surprised that deletion of p28 did not translate into an increase in Tax activity or p19 Gag expression Although p28 mRNA is easily detectable following transient transfection with proviral clones (Fig 2C), we have been unable to detect p28 protein by Western blot [30] (Fig 2C and data not shown) We then determined the effect of exogenously over-expressed p28- and Δp28-AU1 tagged proteins on Tax-mediated transcription Our results confirmed previous reports that over-expressed p28 from a CMV-cDNA expression vector significantly repressed Tax activity in a dose-dependent manner (Fig 3A) Importantly, the Δp28 cDNA expression vector failed to repress Tax activity (Fig 3A) Western blot analysis confirmed the expression of p28 and that the Δp28 amino terminal truncation mutation resulted in a complete loss of p28 protein expression (Fig 3B) Therefore, our results are consistent with the conclusion that either p28 protein is not expressed from the proviral clone following transient transfection (48 hours) or that the levels of p28 expressed from the proviral clone are below the threshold concentration required for detection by Western blot and necessary for repression of Tax or Rex activity 6Kb 7Kb 8Kb 3’LTR pol Structural & enzymatic proteins pro env rex tax p28 rex p22/20 p10 Regulatory proteins Accessory proteins p11 Figure Organization of the HTLV-2 genome and coding regions Organization of the HTLV-2 genome and coding regions The complete proviral genome is shown schematically Boxes denote long terminal repeats (LTRs) RNAs encoding the various protein ORFs are indicated p28 has the potential to be encoded by two distinct singly-spliced mRNAs (gray line in p28 mRNA denotes utilization of two distinct splice acceptor sites) The arrow above p28 ORF depicts the location of the termination mutation to generate Δp28 (stop codon at amino acid 7) Page of 11 (page number not for citation purposes) Retrovirology 2008, 5:38 http://www.retrovirology.com/content/5/1/38 A A 24000 18000 16000 20000 14000 16000 RLU RLU 12000 10000 8000 12000 6000 8000 4000 4000 2000 p19 pg/ml B BC12 HTLV-2 p28 HTLV-2 100 + - + 0.2 - + 0.4 - + 0.2 + 0.4 80 B 60 40 20 C BC12 p28 -actin HTLV-2 HTLV-2 104 103 102 101 100 C p28 105 copy # per 106 gapdh - gag/pol tax/rex mRNA transcript p28 Figure Characterization of proviral clones in vitro Characterization of proviral clones in vitro 293 T cells (2 × 105) were co-transfected with μg of wtHTLV-2 or HTLV-2Δp28 proviral clones or negative control DNA along with 0.1 μg of LTR-1-Luc and 0.01 μg of TK-Renilla All transfections were performed in triplicate and normalized to TK-Renilla to control for transfection efficiency Cell lysates or supernatants were harvested 48 h post-transfection (A) Measure of Tax activity presented as relative luciferase units Results indicated that loss of p28 expression from the proviral clone did not significantly alter Tax activity (B) Rex activity as measured by expression of p19 Gag (virions) in the cellular supernatants Results indicated that loss of p28 expression from the proviral clone did not significantly alter Rex activity (C) Total RNA was extracted from 293 T cells transfected with HTLV-2 or HTLV-2pΔ28 as in panels A and B mRNA copy number was quantified by Taqman realtime RT-PCR The histogram represents the copy number of gag-pol, tax/ rex, and p28 transcripts normalized to × 106 copies of gapdh Results indicated that deletion of p28 protein had no significant affect on tax/rex, gag/pol, or p28 mRNA expression Figure expressed p28, but not Δp28 results in dependent repression of Tax-mediated transcription doseExogenously Exogenously expressed p28, but not Δp28 results in dose-dependent repression of Tax-mediated transcription 293 T cells (2 × 105) were co-transfected with μg of wtHTLV-2 proviral clone or negative control DNA, 0.1 μg of LTR-2-Luc and 0.01 μg of TK-Renilla, and varying concentrations (0.2–0.4 μg) of CMVp28AU1 or CMVΔp28AU1 expression vectors as indicated (A) Tax function was measured as firefly luciferase activity from LTR-2-Luc normalized to Renilla luciferase activity RLU, relative light units (B) Western blot analysis was performed on lysates to confirm expression of p28 (AU1 antibody) or β-actin as a loading control As expected, results indicated that the Δp28 mutation disrupts p28 protein expression HTLV-2Δp28 promoted virus-induced proliferation and immortalization of PBMCs To determine the capacity of HTLV-2Δp28 to synthesize viral proteins, direct viral replication, and induce cellular immortalization, stable 729 cell transfectants expressing wild-type and p28-deleted HTLV-2 proviral clones were generated and characterized Four independent stable HTLV-2Δp28 transfectants were isolated and found to contain complete copies of the provirus; the presence of the expected Δp28 mutation was confirmed by sequencing (data not shown) We quantified the concentration of p19 Gag produced in the culture supernatant of the four cell clones by ELISA Our results showed p19 Gag expression ranging from 250–750 pg/ml (Fig 4A) The variable Page of 11 (page number not for citation purposes) Retrovirology 2008, 5:38 A http://www.retrovirology.com/content/5/1/38 900 800 600 500 400 Clone 100 Clone 200 Clone 300 Clone p19 (pg/ml) 700 729 B 729 729 HTLV-2 729 HTLV-2 729.HTLV-2 Tax-2 -actin 1.7E+0 6.5E+3 2.1E+3 } Copies p28 mRNA Figure permanent of p19 Gag and Expression transfectants Tax protein and p28 mRNA in Expression of p19 Gag and Tax protein and p28 mRNA in permanent transfectants (A) Four 729 stable transfectants (clone 1–4) were isolated for HTLV-2Δp28 as described in Materials and Methods Our well-established 729pH6neo (729.HTLV-2) cell clone was used as the wtHTLV-2 stable producer cell line p19 Gag was quantified by ELISA from the four independently isolated 729.HTLV-2Δp28 (Clones 1–4), 729.HTLV-2, and the 729 negative control Each 729.HTLV-2 producer cell line displayed variable p19 production (B) Clones indicated by asterisks, which have been shown to produce similar quantities of p19 Gag, were further characterized by Western blot for Tax protein expression using rabbit polyclonal antisera raised against Tax-2 β-actin was used as a loading control Numbers below each lane are the copy number of p28 transcript per 106 copies of GAPDH determined by realtime RT PCR The results show similar levels of p28 mRNA expression p19 Gag expression from independent stable cell clones was attributed to chromosomal location of proviral sequences and overall proviral copy number, consistent with previous analyses [44,45] We did not observe a pattern of increased viral gene expression in the absence of p28 For additional studies, we selected 729.HTLV- 2Δp28Clone 3, a stable producer line with p19 Gag production similar to that of our well-characterized wild-type HTLV-2-producer cell line 729pH6neo (729.HTLV-2) Further characterization revealed that, as with transient transfection, p28 mRNA was detected at similar levels (approximately 103 copies per 106 copies of cellular gapdh mRNA) in 729.HTLV-2 and 729.HTLV-2Δp28Clone (Fig 4B), but Western blot analyses failed to detect p28 protein in 729.HTLV-2 or 729.HTLV-2Δp28 (data not shown) A similar level of Tax-2 expression in 729.HTLV2 and 729.HTLV-2Δp28 relative to the β-actin loading control was detected by Western blot and, as expected, Tax-2 was not detected in the 729 negative control cells (Fig 4B) Therefore, as with transient transfection, the repressive effect of p28 expressed from a stably integrated provirus on Tax-mediated transcription was not detectable We assessed the ability of the HTLV-2Δp28 to induce proliferation and immortalize human PBMCs in co-culture assays Freshly isolated human PBMCs co-cultured with lethally irradiated 729.HTLV-2 or 729.HTLV-2Δp28 in the presence of 10 U/ml of human IL-2 showed very similar progressive growth patterns consistent with the HTLV-2 immortalization process, whereas control cells died within the first few weeks (Fig 5A) Immortalized PBMCs expressed similar levels of p19 Gag and harbored the expected HTLV-2 sequences indicating that viral transmission was responsible for the immortalization of PBMCs (data not shown) In an effort to obtain a more quantitative measure of the ability of these viruses to infect and immortalize PBMCs, a fixed number of PBMCs were cocultured with virus-producing cells in a 96-well plate assay [45] Since this assay is very stringent as a result of diluting the cultures 1:3 weekly, slowly growing or non-dividing cells are eliminated very quickly and the percentage of surviving wells is an accurate measure of the immortalization efficiency of viruses A Kaplan-Meier plot of HTLV-2induced T-cell proliferation or survival indicated that the percentage of wells containing proliferating lymphocytes was similar between HTLV-2 and two independently isolated HTLV-2Δp28 clones (Fig 5B) Taken together, our results are consistent with the conclusion that p28 is not required for efficient infectivity or HTLV-2-mediated immortalization of primary human T-lymphocytes in culture In vivo rabbit inoculation results To evaluate the role of p28 in vivo, we compared the abilities of 729, 729.HTLV-2, or 729.HTLV-2Δp28 cell lines to transmit virus to rabbits, which is an established model to investigate HTLV infection and persistence [46] Rabbits were inoculated with lethally irradiated cell lines (cell inocula were equilibrated based on their p19 Gag production) and on weeks 0, 1, 2, 4, 6, 8, and 11, whole blood Page of 11 (page number not for citation purposes) Retrovirology 2008, 5:38 A http://www.retrovirology.com/content/5/1/38 80 729 HTLV-2 Cell Number (x105) 70 60 50 40 30 20 10 10 11 12 14 Week B 100 % Surviving Wells 80 C HTLV-2 60 40 20 0 Weeks p28 is dispensable for HTLV-2-mediated proliferation and immortalization of primary T-lymphocytes Figure p28 is dispensable for HTLV-2-mediated proliferation and immortalization of primary T-lymphocytes (A) Human PBMCs were isolated by Ficoll/Paque and co-cultivated with irradiated (10,000 rads) 729, 729.HTLV-2, or 729.HTLV2Δp28 stable cell lines PBMCs (2 × 106) were cultured with irradiated donor cells (1 × 106) in 24 well plates as indicated A representative growth curve of HTLV-2 infected cells is shown Cell viability was determined weekly by trypan blue exclusion (0–14 wks post co-cultivation) The mean and standard deviation of each time point was determined from three independent samples (B) Pre-stimulated PBMCs (104) were co-cultured with × 103 irradiated 729 stable producer cells in 96 well plates The percentages of proliferating wells were plotted as a function of time (wks) Representative Kaplan-Meir plots for wtHTLV2, HTLV-2Δp28, and 729 uninfected control cells are shown Results indicated that the percentage of wells containing proliferating lymphocytes was similar between wtHTLV-2 and HTLV-2Δp28 infected cells was collected and processed for isolation of plasma and PBMCs Antibody response to viral antigens was detectable by Western blot in all rabbits inoculated with cells expressing either wild type HTLV-2 or HTLV-2Δp28, and the antibody titers in the majority of the rabbits increased over the time course of the study (data not shown) Moreover, quantitative comparison of antibody responses between each rabbit was performed using an HTLV-specific ELISA (Fig 6) Statistical analysis of titers at six, eight, and eleven weeks post-inoculation revealed a significantly lower antibody response to HTLV-2 antigens in the 729.HTLV-2Δp28-inoculated rabbits as compared to the wild-type HTLV-2 control group Consistent with our antibody data, HTLV-2 proviral DNA sequences were detected in all wild type HTLV-2 and five of six HTLV-2Δp28infected rabbits at two weeks post inoculation (Table 1) However, over time, HTLV-2Δp28 failed to persist and quantitative real-time Taqman PCR revealed that at eleven weeks post inoculation, proviral loads in rabbits infected with HTLV-2Δp28 were below the level of detection Page of 11 (page number not for citation purposes) Retrovirology 2008, 5:38 http://www.retrovirology.com/content/5/1/38 1.2 O.D 0.8 0.6 0.4 0.2 Wks 11 729 11 11 729.HTLV-2 Figure Assessment of HTLV-2 infection in rabbits Assessment of HTLV-2 infection in rabbits Antibody response against HTLV-2 from each rabbit was measured by antiHTLV commercial ELISA assay, using both HTLV Gag and envelope proteins as antigens Each dot represents the absorbance value of a single inoculated rabbit at 0, 2, 4, 6, 8, and 11 wks post inoculation within each group Inocula used for the rabbits were 729.HTLV-2 (n = 6), 729.HTLV-2Δp28 (n = 6), or 729 (n = 2) The horizontal line represents the average of the rabbit group at each weekly time point and the dotted line represents three times the standard deviation of uninfected control values Taken together, our results indicated that p28, while dispensable for HTLV-2 infection, attenuated virus replication as measured by antibody response to viral antigens and proviral loads This attenuation was apparent within two weeks post inoculation, suggesting that p28 is required early for efficient replication and survival in the host Discussion The importance of the HTLV-2 nonstructural or accessory proteins in virus biology either in cell culture or in inoculated animals has not been investigated thoroughly A previous study evaluated an HTLV-2 molecular clone containing a large deletion within the proximal pX region, which at the time was thought to delete the coding sequences for all the known accessory proteins Results from this study indicated that this region, which later was shown to contain open reading frames (ORFs) for p10 and p11 [28], was dispensable for viral infection and cellular transformation in vitro [41] Subsequently, it was demonstrated that this deletion resulted in reduced proviral load and maintenance of infection in vivo [42] However, the role of the HTLV-2 p28 accessory protein encoded by ORF II located in exon of tax/rex was not addressed directly in these studies We previously demonstrated that exogenously over-expressed p28 functions as a negative regulator of viral replication by binding to and retaining tax/rex mRNA in the nucleus, thus repressing Tax and Rex protein production and overall viral gene expression [30,31] In this study, a site directed mutation was introduced in an infectious clone of HTLV-2 that severely truncated p28 (HTLV-2Δp28) while maintaining the ability of the virus to express other gene products Subsequently, we examined the expression of p28 and determined its biological significance for the infectivity and immortalization of primary T-lymphocytes in cell culture and viral infectivity and persistence in vivo Data from our transient transfection studies revealed that, in the context of a proviral clone, the repressive effects of p28 on Tax-mediated transcription and Rex function were not apparent (Fig 2A &2B) In fact, the loss of p28 resulted in a reproducible, but not significant decrease in Tax activity (75–90%) Consistent with the functional reporter assays, quantitative real-time RT-PCR revealed that the levels of tax/rex and gag/pol mRNA were not dramatically different in cells transfected with HTLV-2 and HTLV-2Δp28 proviral clones (Fig 2C) Although we could detect p28 encoding mRNA (approximately 103-104 total copies per 106 copies of gapdh), p28 protein was below the limit of detection by Western blot Due to alternative splicing, p28 has the potential to be expressed from two distinct singly-spliced mRNAs (both of these mRNAs also have the potential to produce the truncated p22/p20rex) Studies by Li and Green showed that these two mRNAs have significantly different expression levels in newly infected PBMCs (105 vs 103 copies per 106 copies of cellular gapdh) [43] Although nearly impossible to definitively confirm experimentally, we hypothesize that the low copy number mRNA is the primary transcript utilized to encode p28, thus resulting in low protein expression (below our limit of detection) To date, with the exception Page of 11 (page number not for citation purposes) Retrovirology 2008, 5:38 http://www.retrovirology.com/content/5/1/38 Table 1: Detection of HTLV-2 sequences in PBMCs from inoculated rabbitsa Weeks Post Inoculation Inoculum and Rabbit 729.HTLV-2 R27 R28 R29 R30 R31 R32 729.HTLV-2Δp28 R20 R21 R22 R23 R24 R25 729 R1 R6 11b - + + + + + + + + + + + + + + + +/- + (12.0) + (8.3) + (5.3) + (32.8) + (10.7) + (4.2) - + +/+/+/+/- +/- + - - (0.2) - (0.1) - (0.3) - (0.3) - (1.1) - (1.2) - - - - - (0.1) - (0.3) aGenomic DNA was isolated from rabbit PBMCs and subjected to standard PCR (40 cycles) using HTLV-2 specific primers (TRE-pH6-S/ TRE-pH6-AS) -, no amplified PCR fragment; +, amplified PCR fragment bNumbers in parentheses at wk 11 denote copy number per 1000 cells of rabbit PBMC as determined by real-time RT PCR Copy numbers in rabbits inoculated with 729.HTLV-2Δp28 at wk 11 were significantly different than 729.HTLV-2 as determined by ANOVA followed by Turkey's test (p